WO2018160090A1 - Antiviral composition and method for using same - Google Patents

Abstract

The present invention relates to an antiviral composition and a method for using same to treat the hepatitis C virus in patients. An antiviral composition comprising the HCV NS5B inhibitor of general formula 1 or a stereoisomer thereof, an isotopically enriched analogue, a crystalline or polycrystalline form.

Description

 Antiviral composition and method of its use

Technical field

 Hepatitis C caused by hepatitis C virus (HCV) is one of the most common liver diseases and is widespread throughout the world. Based on the annual reports of the World Health Organization (WHO), more than 130-150 million people are infected with HCV and more than 700 thousand people die from HCV [WHO. Hepatitis C. WHO fact sheet N ° 164. Updated July 2016, http: // www, who. i nt / medi acentre / factsheets / fsl64 / en /]. HCV has a high genetic diversity and is characterized by regional variations in the HCV genotypes (gT). Genotype 1 (gTl) is the most common worldwide (83.4 million people, accounting for 46.2% of all cases of HCV infection), approximately one third of which are in East Asia. The next most common is genotype 3 (gT3). 54.3 million people (30, 1%) are infected with this genotype worldwide. Genotypes 2, 4, 6 make up 22.8% of all cases of HCV-infected humans, and genotype 5 (gT5) makes up the remaining <1%. While genotypes 1 and 3 dominate in most countries, regardless of economic status, the largest fractions of genotypes 4 and 5 are in low-income countries [Messina, J. P. at al. Global Distribution and Prevalence of Hepatitis With Virus Genotypes. Hepatology 2015, 61 (1), 77-87.].

State of the art

Significant progress in the treatment of hepatitis C has been achieved in recent years, which is primarily associated with the discovery of Sofosbuvir (Sofosbuvir, Sovaldi, PSI-7977, GS-7977), an HCV NS5B nucleoside inhibitor [Sofia, MJ et al. Discovery of a β-ϋ-20-Deoxy-20-rfluoro-2 Ο-β-C-methyluridine Nucleotide Prodrug (PSI-7977) for the Treatment of Hepatitis C Virus. J. Med. Chem. 2010, 53, 7202-7218. Sofia, MJ et al. Nucleoside phosphoramidate prodrugs. Patent US 7964580, 201 1. Sofia, MJ Nucleoside phosphoramidate prodrugs. Patent US 8334270, 2012.] and Daclatasvir (Daclatasvir, BMS-790052), an inhibitor of the inhibitor of NS5A HCV [Belema, M. et al. Discovery and Development of Hepatitis With Virus NS5A Replication Complex Inhibitors. J. Med. Chem. 2014, 57, 1643-1672. Bachand, C. et al. Hepatitis C virus inhibitors. Patent WO 2008/021927, 2008. Bachand, C. et al. Hepatitis with virus inhibitors. Patent WO 2008/021928, 2008. Bachand, C. et al. Hepatitis C virus inhibitors. Patent WO2008 / 021936, 2008.

 http://www.ema.europa.eu/docs/en_GB/document_library/EP AR_Public_assessment report human / 003768 / WC500172849. df.].

 Sovaldi (GS-7977, PSI-7977, Sofosbuvir)

 Declatasvir (BMS-790052)

Other NS5A inhibitors, including Ledipasvir (Ledipasvir, GS-5885) [Link, J. O. et al. Discovery of ledipasvir (GS-5885): a potent, once-daily oral NS5A inhibitor for the treatment of hepatitis C virus infection J. Med. Chem. 2014, 57, 2033-2046. Guo, H. et al. Antiviral compounds. Patent WO 2010/132601, 2010. http://www.ema.europa.eu/docs/en_ GB / document_library / EPAR_Public_assessment_ report / human / 003850 / WC500177996.pdf. http://www.hcvdruginfo.ca/downloads/HCV_ ledipasvir.pdf],

 Ombitasvir (Ombitasvir, ABT-267) [Gardelli, C. et al. Phosphoramidate Prodrugs of 20-C- Methylcytidine for Therapy of Hepatitis C Virus Infection. J. Med. Chem. 2014, 57, 2047-2057. Patent WO 2010/144646, 2010.],

 Ombitasvir (ABT-267)

Elbasvir (Elbasvir, MK-8742) [Coburn, CA et al. ChemMedChem. 2013, 8, 1930-1940. Patent WO 2012/040923, 2012. Patent WO 2012/041014, 2012],

 Elbasvir (MK-8742)

Velapatasvir (Velpatasvir, VEL, GS-5816) [Patent WO 2015/1 10048, 2015. http://www.accessdata.fda.gov/ drugsatfdadocs / nda / 2016/2083410 riglsOOOPharmR.pdf]. http: // www. ilead.eom / ~ / media / files / pdfs / medicines / liver-disease / epclusa / epclusa_pi.pdf |.

Hepavivir (Hepavivir, AV-4025) Ivachtchenko, AV et al. Discovery of Novel Highly Potent Hepatitis With Virus NS5A Inhibitor (AV4025). J. Med. Chem. 2014, 57, 7716-7730. Patent WO 2012/074437, 2012. Patent US 9428491, 2016.], AV-4056 and AV-4058 [US 9428491.], AV-4067 and AV-4084 [Pat. Appl. US 14 / 845,333.],

 AV-4067

Samagasvir (Samatasvir, XX 18719, IDX 719) [Patent WO 201 1075615, 201 1. Bilello J. P. et al. In vitro activity and resistance profile of samatasvir, a novel NS5A replication inhibitor of hepatitis C virus. Antimicrobobial Agents Chemother. 2014, 58 (8), 4431-4442.],

 Samatasvir (IDX 18719, IDX 719)

Odalasvir (ACH-3102) [Nakamoto S. et al. Hepatitis C virus NS5A inhibitors and drug resistance mutations. World J. Gastroenterol. 2014, 20 (1 1), 2902-2912.].

 Odalasvir (ACH-3102)

 The combination of NS5A and NS5B inhibitors allows not only to exclude the use of interferon and ribavirin for the treatment of hepatitis C, but also to radically increase the effectiveness of treatment. For example, Clinical trials of the combined drug Epclusa (Epclusa tablet contains 400 mg Sofosbuvir and 100 mg Velapatasvir) showed high efficacy (SVR12> 95%) in subjects without cirrhosis and in subjects with compensated cirrhosis genotype 1, 2, 3, 4, 5 and 6 HCV infections in adults [http: //vvww.gilead.eom/~/media files / pdfs / medicines / Uver-dis epclusa / epclusa_pi.pdfj.

To date, protease inhibitors have also been used successfully in combination therapy for hepatitis C, including Narlaprevir (Narlaprevir, SCH 900518), an inhibitor of unstructured HCV 3 protein (NS3 HCV) [Arasappan A, et al. Discovery of Narlaprevir (SCH 900518): A Potent, Second Generation HCV NS3 Serine Protease Inhibitor. ACS Med. Chem. Lett., 2010, 1 (2), 64-69] and Simeprevir (Simeprevir, Olysio) an inhibitor of the unstructured protein NS3 / NS4 HCV (NS3 / 4A HCV) [https://www.tga.gov.au/ sites / default /files/auspar-simeprevir-141027-pi.docx].

 Narlaprevir (SCH 900518)

 Simeprevir (Olysio)

Recently, an antiviral composition has been proposed containing, as a prodrug, an NS5B HCV polymerase inhibitor, the PSI-7851 phosphoramidate nucleotide, which is isopropyl (8) -2- {[(2K, 3K, 5K) -5- (2,4-dioxo -3,4-dihydro-2H-pyrimidin-1-yl) -4-fluoro-3-hydroxy-4-methyl-tetrahydrofuran-2-ylmethoxy] phenoxyphosphorylamino} propionate or its phosphorus stereoisomers (Rp isomer: PSI -7976 and Sp isomer: PSI-7977) [Patent US 7964580 (201 1), RU 2478104 (2013)].

S _p isomer: PSl-7977 (Sovaldi)

An antiviral composition comprising PSl-7977, known as Sovaldi ^® and Sofosbuvir http://www.gilead.eom/~/media/Files/ pdfs / medicines / liver-disease / sovaldi / sovaldi_pi.pdf] is currently widely used for combined treatment of hepatitis C, including, together with NS5A inhibitors HCV, NS3, NS3 / 4 and other antiviral components. PSI-7851 and its stereoisomers PSI-7976 and PSI-7977 are metabolized to triphosphate PSI-7409, which is an inhibitor of NS5B HCV polymerase [E. Murakami et al. Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977. J Biol. Chem. 2010, 285 (45), 34337-34347].

The drug Sovaldi ^® [M. J. Sofia et al. Discovery of a pD-20-Deoxy-20-r-fluoro-20-PC- methyluridine Nucleotide Prodrug (PSI-7977) for the Treatment of Hepatitis C Virus. J. Med. Chem. 2010, 53, 7202-7218. MJ Sofia et al. Nucleoside phosphoramidate prodrugs. US 7964580 (2011).] Became the first nucleotide approved by both regulatory authorities of the FDA and the EU for the combined treatment of hepatitis C patients infected with different genotypes (gT) of hepatitis C. In clinical trials, it showed high efficacy against six HCV genotypes (gTl-gT6 ) [IM Jacobson et al. Sofosbuvir for hepatitis C genotype 2 or 3 in patients without treatment options. Engl. J. Med. 2013, 368, 1867-1877. E. Lewirz et al. Sofosbuvir for previously untreated chronic hepatitis C infection. Engl. J. Med. 2013, 368, 1878-1887].

 Despite the progress made in recent years in the treatment of hepatitis C, the challenge remains of finding new antiviral compositions that include a prodrug of HCV NS5B inhibitors, with a more efficient prodrug metabolism to PSI-7409 triphosphate and improved properties.

Disclosure of invention

The authors unexpectedly found that antiviral compositions containing, as a prodrug of an NS5B HCV polymerase inhibitor, a previously unknown compound of the general formula 1 or its phosphorus stereoisomer of formula 1.1 or 1.2 (Sp stereoisomer: 1.1 and Rp stereoisomer: 1.2) are an effective antiviral agent, including for the treatment of hepatitis C

The inventors found that the total concentration of PSI-7409 triphosphate formed as a result of metabolism in the liver of rats during the entire time of observation of a previously unknown prodrug of cyclobutyl (5) -2 - {(5) - [(2i?, 3i?, 4i?, 5i? ) -5- (3,4-dihydro-2,4-dioxo-2 ^ pyrimidin-1-yl) -3-hydroxy-4-methyl-4-fluoro-tetrahydro-furan-2-ylmethoxy] -phenoxy-phosphorylamino } propanoate (1.1) has a value AUCo- _t = 27079 hr ng / g, which is 1.6 times higher than the total concentration of PSI-7409 (AUCo _-t = 16796 ng hr / g) of the resulting Sovaldi ^® metabolism in rat liver [ M. J. Sofia et al. Discovery of a pD-20-Deoxy-20-r-fluoro-20- -C-methyluridine Nucleotide Prodrug (PSI-7977) for the Treatment of Hepatitis C Virus. J. Med. Chem. 2010, 53, 7202-7218].

Note that currently in the combined treatment of hepatitis C, an antiviral composition in the form of tablets is used, including 400 mg of Sovaldi ^® , which is the most effective daily dose. New antiviral the composition is more effective in the treatment of hepatitis C, because it includes an active component, in particular, a prodrug of formula 1.1, which is more efficiently metabolized in the liver of a mammal to triphosphate PSI-7409, which is an inhibitor of NS5B HCV polymerase. This allows you to reduce the daily dose of the prodrug of formula 1.1 in comparison with the dose of Sovaldi ^® . The latter, in turn, will reduce side effects.

 The above data convincingly confirm the novelty of the invention and its inventive step (efficiency).

 The following are definitions of various terms used to describe the present invention. These definitions apply to the terms as they are used in this description and the claims, unless otherwise limited in specific cases, either individually or as part of a larger group.

 The term “active component” (drug substance) refers to a physiologically active substance of a synthetic or other (biotechnological, plant, animal, bactericidal, etc.) origin having pharmacological activity, which is an active ingredient in a pharmaceutical composition.

 The term "crystalline form" is the structure of a substance, characterized by the packing of the molecules that form them into one of the types of crystal lattice.

 The term "polycrystalline form" is a structure of a substance having a polycrystalline structure, i.e. consisting of many small single crystals, i.e. crystallites of a certain crystalline form.

 The term “drug” means a substance (or a mixture of substances in the form of a pharmaceutical composition) in the form of tablets, capsules, injections, ointments, and other formulations intended to restore, correct, or alter physiological functions in humans and animals, as well as for the treatment and disease prevention, diagnosis, anesthesia, contraception, cosmetology and more.

The term "pharmaceutical composition" means a composition comprising a compound of general formula 1 and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible fillers, solvents, diluents, carriers, auxiliary, dispensing and perceptive means, delivery vehicles, such as preservatives, stabilizers, fillers, grinders, moisturizers, emulsifiers, suspending agents, thickeners, sweeteners, perfumes, flavors, antibacterial agents, fungicides, lubricants, regulators of prolonged delivery, the choice and ratio of which depends on the nature and method of administration and dosage. Examples of suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitol ether, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, as well as mixtures of these substances. Protection against the action of microorganisms can be provided with a variety of antibacterial and antifungal agents, for example, parabens, chlorobutanol, sorbic acid and the like. The composition may also include isotonic agents, for example, sugars, sodium chloride and the like. The prolonged action of the composition can be achieved using agents that slow down the absorption of the active principle, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols, and also mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Examples of excipients are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate and the like. Examples of grinders and distributors are starch, alginic acid and its salts, silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol. The pharmaceutical composition for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, local or rectal administration of the active principle, alone or in combination with another active principle, can be administered to animals and humans in a standard administration form, in the form of a mixture with traditional pharmaceutical carriers. Suitable unit dosage forms include oral forms such as tablets, gelatine capsules, pills, powders, granules, chewing gums and oral solutions or suspensions, sublingual and buccal administration forms, aerosols, implants, local, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular forms of administration and rectal forms of administration.

 The term “inert excipient”, as used herein, refers to a compound that is used to produce a pharmaceutical composition and is generally safe, non-toxic, neither biologically nor otherwise undesirable, and includes excipients that are acceptable for veterinary use, as well as pharmacologically acceptable for human use. The compounds of this invention may be administered separately, but will usually be administered in a mixture with one or more pharmaceutically acceptable excipients, diluents or carriers selected according to the intended route of administration and standard pharmaceutical practice.

 The term “therapeutically effective amount” as used herein means the amount of a substance, prodrug, or drug necessary to reduce the symptoms of a disease in a subject. The dose of a substance, prodrug or drug will meet the individual requirements in each case. This dose can vary widely depending on many factors, such as the severity of the disease to be treated, the age and general health of the patient, other drugs with which the patient is being treated, the method and form of administration and the experience of the attending physician. For oral administration, the daily dose is from about 0.01 to 10 g, including all values between them, per day, in monotherapy and / or in combination therapy. A preferred daily dose is from about 0.1 to 7 g per day. Typically, treatment is started with a large initial “loading dose” to quickly reduce or eliminate the virus accompanying the decreasing dose to a level sufficient to prevent an outbreak of infection.

The term “subject” means a mammal that includes, but is not limited to, cattle, pigs, sheep, chicken, turkeys, buffalos, llamas, ostriches, dogs, cats, and humans, preferably the subject is a human. It is contemplated that in the method of treating a subject, any of the prodrugs may be common formula 1, its stereoisomers, isotopically enriched analogs, its pharmaceutically acceptable salts, hydrates, solvates, crystalline and polymorphic forms, or in combination with another compound, including an NS5A HCV inhibitor.

 The subject of this invention is an antiviral pharmaceutical composition containing, as a prodrug, an inhibitor of the NS5B HCV polymerase cyclobutyl (5 -2- {[(2 g, 3 g, 4 g, 5L) -5- (2,4-dioxo-3,4- dihydro-2H-pyrimidin-1-yl) -4-fluoro-3-hydroxy-4-methyl-tetrahydrofuran-2-ylmethoxy] phenoxyphosphorylamino} propanoate of the general formula 1, and / or its phosphorus stereoisomer (Sp stereoisomer: formulas 1.1 or i? p stereoisomer: formulas 1.2), their isotope-rich analogue, crystalline or polycrystalline form.

 A preferred antiviral pharmaceutical composition comprises, as a prodrug, an inhibitor of NS5B HCV polybase cyclobutyl (S) -2 - {(S) - [(2K, ZK, 4K, 5K) -5- (3,4-dihydro-2,4-dioxo 2 ^ pyrimidin-1-yl) -3-hydroxy-4-methyl-4-fluoro-tetrahydro-furan-2-ylmethoxy] -phenoxy-phosphorylamino} -propanoate of formula 1.1, its isotopically enriched analogue, crystalline or polycrystalline form.

The new antiviral pharmaceutical composition, as shown above, is more effective than known compositions, including containing Sovaldi ^® as an inhibitor of NS5B HC V polymerase

 A subject of the present invention is an antiviral pharmaceutical composition for treating hepatitis C virus in mammals, comprising a prodrug of general formula 1 or a stereoisomer thereof, an isotope-enriched analogue, crystalline or polycrystalline form, optionally in combination with a pharmaceutically acceptable excipient, carrier, additive or diluent.

The antiviral pharmaceutical composition may be in the form of a wide variety of orally administered dosage forms and carriers, including in the form of tablets, coated tablets, hard and soft gelatin capsules, solutions, emulsions, syrup or suspension. The antiviral pharmaceutical composition of the present invention is effective when administered as a suppository. The most convenient route of administration is usually oral using the usual daily dosage regimen, which can be adjusted to depending on the severity of the disease and the patient's response to the antiviral and antitumor drug.

 An antiviral pharmaceutical composition with one or more conventional excipients, carriers or diluents may be presented in the form of pharmaceutical compositions and unit doses. The pharmaceutical compositions and unit dosage forms may be comprised of the usual ingredients in the usual proportions with or without additional active compounds and dosage forms. The antiviral composition may contain any appropriate effective amount of the active ingredient commensurate with the prescribed daily dose. The antiviral composition may be used in the form of solids, such as tablets or filled capsules, in the form of semi-solid powders, sustained release preparations or liquids, such as suspensions, emulsions or filled capsules for oral administration; or in the form of suppositories for rectal or vaginal administration. A typical preparation will contain from about 5% weight. up to 95% weight. active compound or compound. The term “preparation” or “dosage form” is intended to include both solid and liquid compositions of the active compound, and one skilled in the art will understand that the active ingredient may exist in the form of various preparations depending on the required dose and pharmacokinetic parameters.

Solid form preparations include powders, tablets, pills, capsules, suppositories, and dispersible granules. A solid carrier may be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents or encapsulating material. In powders, the carrier is usually a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component is usually mixed with a carrier having the necessary binding ability, in suitable proportions, and compressed into the desired shape of the desired size. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting wax, cocoa butter and the like. Solid form preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents and the like.

 Liquid formulations are also suitable for oral administration. Liquid dosage forms are emulsions, syrups, elixirs and aqueous suspensions. These include solid form preparations which are intended to be converted to liquid preparations immediately prior to use. Emulsions can be prepared in solutions, for example, in aqueous solutions of propylene glycol or may contain emulsifiers such as lecithin, sorbitan monooleate or gum arabic. Aqueous suspensions may be prepared by dispersing the finely divided active component in water with viscous materials such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose and other well-known suspending agents.

 An antiviral pharmaceutical composition may be prepared for administration in the form of suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously, for example by stirring. The molten homogeneous mixture is then poured into molds of a convenient size, allowed to cool and harden.

 An antiviral pharmaceutical composition may be prepared for vaginal administration. The use of suppositories, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, such carriers as are known in the art will be appropriate.

More preferred is an antiviral pharmaceutical composition, which together with a new prodrug of general formula 1 or a stereoisomer of formula 1.1, an isotopically enriched analogue, crystalline or polycrystalline form, further comprises an antiviral or anticancer agent in a therapeutically effective amount. More preferred is an antiviral pharmaceutical composition, which, together with a new prodrug of general formula 1 or a stereoisomer of formula 1.1, an isotopically enriched analogue, crystalline or polycrystalline form, further comprises a therapeutically effective amount of an HCV NS5A inhibitor selected from the group consisting of Daclatasvir (Daklinza) , BMS790052) Hepavivir (AV-4025), AV-4056 and AV-4058, Ombitasvir (AVT-267), Elbasvir (MK-8742) or Velpatasvir (Velpatasvir) (VEL, GS-5816) or Narlaprevir (Narlaprevir, SCH 900518) - n inhibitor Structured HCV protein 3 (NS3 HCV or Simeprevir (Simeprevir, Olysio) inhibitor unstructured protein NS3 / NS4 HCV.

 An antiviral pharmaceutical composition which, together with a new prodrug of general formula 1 or a stereoisomer of formula 1.1, an isotope-enriched analogue, crystalline or polycrystalline form, further comprises a therapeutically effective amount of an HBV DNA polymerase inhibitor and an HIV-1 reverse transcriptase (RT) inhibitor, is also more preferred. .

 The subject of the present invention is a method for the combined treatment of viral and cancer diseases in a subject in need thereof, said method comprising the sequential or simultaneous administration of a therapeutically effective amount of an antiviral pharmaceutical composition comprising a prodrug of general formula 1 or its stereoisomer of formula 1.1, an isotopically enriched analogue , crystalline or polycrystalline form and other antiviral or anticancer agents. It is clear that the time between separate administration of drugs can be in a range that includes any time range.

The subject of the present invention is a method for the combined treatment and / or prophylaxis of a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of an antiviral pharmaceutical composition containing, as a prodrug, an inhibitor of NS5B HCV polymerase cyclobutyl (.5) -2 - {[(2 ^ ZL, 47g, 5L) -5- (2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl) -4-fluoro-3-hydroxy-4-methyl-tetrahydrofuran-2-ylmethoxy] -phenoxy-phosphorylamino} - propanoate of general formula 1, and / or its phosphorus stereoisomer (5p stereoisomer: formula 1.1 or Rp stereoisomer: formula 1.2), their isotope-rich analogue, crystalline or polycrystalline form, according to this invention, which may include an additional therapeutically effective amount of one or more other antiviral or anticancer agents that use parallel or alternative administration of agents. It is clear that the time between sequential administration of agents can be in any time range.

 Examples of “other antiviral agents” include, but are not limited to: HCV NS3 protease inhibitors [US US 20140296136, US 8,987,195, US 7973040, US 2012214783], NSV HCV inhibitors [EP1497282], HCV NS3 / NS4 inhibitors [EP 2364984] inhibitors of NS5A HCV [C. Wang et al. Hepatitis C virus RNA elimination and development of resistance in replicon cells treated with BMS-790052. Antimicrob. Agents Chemother. 2012, 56, 1350-1358. https://en.wikipedia.org/wiki/Daclatasvir; A.V. Ivachtchenko et al. Discovery of Novel Highly Potent Hepatitis With Virus NS5A Inhibitor (AV4025). J Med Chem. 2014, 57, 7716-7730; Pat. Appl. US 14 / 845,333); Toll-like receptor agonists (see WO 2015023958, WO 2012097012); and other inhibitors (see WO 2014106019, WO 2014033176, WO 2014033170, WO 2014033167, WO 2013006394, US 20090163545].

It is contemplated that another antiviral agent is, but is not limited to: interferon alpha, interferon beta, pegylated interferon alpha, ribavirin, levovirin, viramidine, another nucleoside HCV polymerase inhibitor, non-nucleoside HCV polymerase inhibitor, HCV protease inhibitor, HCV x an inhibitor or HCV fusion inhibitor, an HBV DNA polymerase inhibitor, and an HIV-1 reverse transcriptase (RT) inhibitor. When a prodrug of general formula 1 or its stereoisomer, isotopically enriched analogs, crystalline or polycrystalline forms are administered in combination with another antiviral or anticancer agent, activity can be increased compared to the original prodrug. When the treatment is combined, the administration of drugs can be simultaneous or sequential with respect to a prodrug of general formula 1 or its stereoisomers, isotope-enriched analogs, crystalline or polycrystalline forms. The term "simultaneous administration", as used here, thus, includes the introduction of agents at the same time or at different times. Reception of two or more agents at the same time can be achieved using a single formulation containing two or more active ingredients, or essentially the simultaneous administration of two or more dosage forms with one active agent. It should be understood that the references here to treatment extend to prevention. In addition, the term “treating” a viral infection, as used herein, also includes the treatment or prophylaxis of a disease or condition associated with an indirect viral infection, or their clinical symptoms.

The authors unexpectedly found that a new antiviral pharmaceutical composition, comprising a prodrug of general formula 1 and / or its stereoisomer of formula 1.1, is more effective than the known antiviral composition, including more effective than containing Sovaldi ^® .

In particular, Sovaldi ^® with respect to the lb (gTlb) HCV genotype has an EU _{50 of} 0.045-0.170 μΜ [http://www.hcvdruginfo.ca/downloads/HCV Sofosbuvir.pdfl and an ESED of 0.59 μΜ, and the new prodrug of formula 1.1 has an EU of ₅₀ 15.0-27.0 pM and EC90 128.0 pM (Table 2), i.e. the new prodrug of formula 1.1 is more than three times more active than Sovaldi ^® . The half-life of a prodrug of formula 1.1 in the S9 fraction of human liver microsomes is T ^ ⁹ = 0.05 hours, and in Sovaldi ^®, respectively, T _\ a ^hS9 = 0.57 hours (Table 3), i.e. a new prodrug of formula 1.1 is 1 to 1 times faster metabolized in the S9 fraction of human liver microsomes than Sovaldi ^® . In addition, the concentration and _{24 h} AUC of PSI-7409 triphosphate formed during the metabolism of a prodrug of formula 1.1 in rat liver is Сшах = 3.224.0 ng / g and AUC ₂₄ = 30.487.0 ng h / g, while with a similar metabolism, Sovaldi ^® has Stax = 1.934.0 ng / g and AUC ₂ 4 _h = 16.796.0 ng h / g (Table 4). This suggests that the new prodrug is almost 2 times more efficiently metabolized in the liver to the desired triphosphate PSI-7409 (medicine). The best embodiment of the invention

The present invention will now be described in connection with certain embodiments that are not intended to limit its scope. On the contrary, the present invention covers all alternatives, modifications and equivalents that may be included in the scope of the claims. Thus, the following examples, which include specific embodiments, illustrate but do not limit the present invention.

Example 1. The synthesis protocol of the prodrug cyclobutyl (5) -2 - {[(2?, 3Λ, 4Λ, 5 /?) - 5- (3,4-dihydro-2,4-dioxo-2H-pyrimidin-1-yl ) -3-hydroxy-4-methyl-4-fluoro-tetrahydro-furan-2-ylmethoxy] -phenoxy-phosphorylamino} -propanoate of the general formula (1) and its stereoisomers of the formula 1.1 and the formula 1.2 (Scheme 1).

6

8, 8.1, 8.2

To a solution of N-Boc-L-alanine (4: 15.5 g, 81.9 mmol) in 300 ml of dichloromethane at 0 ° C was added DCC (16.9 g, 81.9 mmol) and after 5 min cyclobutanol (3 : 5.6 g, 78.0 mmol) and DMAP (2.0 g, 16.4 mmol). The mixture was allowed to stir overnight at room temperature, evaporated in vacuo and the residue was taken up in 300 ml of ethyl acetate. The precipitate was filtered off and washed with ethyl acetate. The filtrate was washed with 5% citric acid solution (2 x 100 ml), saturated NaHC0 ₃ solution (2 x 100 ml), brine, dried over Na ₂ S0 ₄ and evaporated in vacuo. 19.6 g (98%) of (5) -cyclobutyl 2- (tert-butoxycarbonylamino) -propanoate (5) are obtained in the form of a white powder. Ή NMR (400 MHz, DMSO-s / ₆ ) δ 7.22 (d, J = 7.2 Hz, 0.85H), 6.87 (m, 0.15H), 4.89 (p, J = 7.2 Hz, 1H), 3.94 (m, 1H), 2.26 (m, 2H), 1.98 (m, 2H), 1.74 (m, 1H), 1.59 (m, 1H), 1.38 (s, 7.5H), 1.34 (brs, 1.5H), 1.22 (d , J = 7.2Hz, 3H).

To a solution of compound 5 (19.6 g, 80.6 mmol) in 50 ml of dioxane was added 230 ml of 3M HCl in dioxane, the mixture was allowed to stir overnight and then evaporated in vacuo. The residue was taken up in 400 ml of ether and allowed to stir overnight. The precipitate was filtered off, washed with ether and dried in vacuo. 14.1 g (97%) of (S) - cyclobutyl 2-amino-propanoate hydrochloride (6) are obtained in the form of a white powder. Ή NMR (400 MHz, DMSO-s / ₆ ) δ 8.56 (brs, 3Н), 5.00 (p, J = 7.6 Hz, 1H), 4.02 (q, J = 7.2 Hz, 1H), 2.31 (m, 2H), 2.07 (m, 2H ), 1.78 (m, 1H), 1.62 (m, 1H), 1.41 (d, J = 7.2 Hz, 3H).

To a solution of compound 6 (14.4 g, 80.2 mmol) in 214 ml of dichloromethane was added phenyl dichlorophosphate (16.9 g, 80.2 mmol). The mixture was cooled to -75-70 ° C and a solution of triethylamine (16.2 g, 160.4 mmol) in 16 ml of dichloromethane was added dropwise, keeping the temperature at -75-70 ° C. The mixture was stirred at -70 ° C for 30 minutes and then heated to -20 ° C. A solution of pentafluorophenol (14.6 g, 79.4 mmol) in 105 ml of dichloromethane at -20-10 ° C is added, then a solution of triethylamine (8.1 g, 80.2 mmol) in 8 ml of dichloromethane is added dropwise and the mixture is left to mix at night at room temperature. The mixture was evaporated in vacuo, 500 ml of ethyl acetate and 500 ml of water were added, the organic layer was separated, washed with 5% NaHC0 ₃ solution, brine, dried over Na ₂ S0 ₄ and evaporated in vacuo. 200 ml of a hexane / ethyl acetate 6: 1 mixture was added to the residue and allowed to stir overnight. The precipitate formed is filtered off, washed with 50 ml of a mixture of hexane / ethyl acetate 6: 1 and dried in air. 16.7 g of cyclobutyl (5) -2- ((perfluorophenoxy) (phenoxy) phosphorylamino) propanoate (2) are obtained.

The resulting product 2 was recrystallized from 500 ml of a hexane / ethyl acetate 4: 1 mixture to obtain 13.8 g (37%) of cyclobutyl (5) -2 - ((5 - (perfluorophenoxy) - (phenoxy) phosphorylamino) propanoate of the formula (2.1) in белого NMR (300 MHz, DMSO- ^ ₆ ) δ 7.42 (m, 2Н), 7.24 (m, ЗН), 6.87 (dd, J, = 14.1 Hz, J ₂ = 10.2 Hz, 1H); 4.87 (p, J = 7.5 Hz, 1H), 3.94 (m, 1H), 2.23 (m, 2H), 1.94 (m, 2H), 1.71 (m, 1H), 1.58 (m, 1H), 1.27 (d , J = 7.2 Hz, ZN). The mother liquor from washing with hexane / ethyl acetate 6: 1 upon recrystallization of the compound of formula 2.1 is evaporated in vacuo and recrystallized from hexane three times. Cyclobutyl (5) -2 - ((??) - (perfluorophene) is obtained xi) - (phenoxy) -phosphorylamino) -propanoate (2.2) as a loose white powder. Ή NMR (300 MHz, DMSO--) δ 7.42 (m, 2Н), 7.26 (m, ЗН), 6.85 (dd, J , = 13.8 Hz, J ₂ = 10.2 Hz, 1H), 4.88 (p, J = 7.5 Hz, 1H), 3.95 (m, 1H), 2.24 (m, 2H), 1.93 (m, 2H), 1.72 (m , 1H), 1.59 (m, 1H), 1.28 (d, J = 6.9 Hz, 3H).

To the solution / wjpem-butyl (2β, 3L, 4L, 5L) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2 / ^) - yl) -2- (hydroxymethyl) -4- methyl 4-fluoro-tetrahydrofuran-3-yl carbonate (7: 5 g, 13.9 mmol) in 165 ml of THF add 1M solution of tert-butylmagnesium chloride in THF (31.3 ml, 31.3 mmol) under argon at 0 ° C and the mixture is stirred for 30 min at room temperature. Then, using a syringe, add a solution of the compound of formula 2.1 (7.8 g, 16.7 mmol) in 30 ml of THF at 0-5 ° C and the mixture is stirred under argon for a day. 10 ml of methanol was added to the mixture and evaporated in vacuo. The residue was dissolved in 500 ml of ethyl acetate, washed with 5% citric acid solution, 5% NaHC0 ₃ solution, dried over Na ₂ S0 ₄ and evaporated in vacuo. The residue was chromatographed on silica gel (eluent hexane / ethyl acetate 1: 2). 5.89 g (66%) of cyclobutyl (5) -2 - ((5} - (((2 ^, 3i?, 4i?, 5i?) - 3- (/? / 7eth-butoxycarbonyloxy) -5- are obtained (2,4-dioxo-3,4-dihydropyrimidin-1 (2 /) -yl) -4-methyl-4-fluoro-tetrahydrofuran-2-yl) methoxy) (phenoxy) phosphorylamino) propanoate (8.1) as a colorless Foam Foam LC-MS (ESI) 642 (M + H) ⁺ .

Cyclobutyl (.S -2 - ((((2i?, 3i?, 4i?, 5R) -3- (wpew-butoxycarbonyloxy) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 ( 2 /) -yl) -4-methyl-4-fluoro-tetrahydrofuran-2-yl) methoxy) (phenoxy) phosphorylamino) propanoate (8). 52% yield. LC-MS (ESI) 642 (M + H) ⁺ , starting from intermediates 7 and 2, and cyclobutyl (iS) -2 - ((?) - (((2ig, 3?, 4?, 5Λ) -3- (like i-bytoxic acid) -5- (2,4 -diocco-3,4-dihydropypyrimidin-l (2H) - yl) -4-methyl-4-fluoro-tetrahydrofuran-2-yl) methoxy) (phenoxy) phosphorylamino) propanoate (8.2) Yield 59%. LC-MS (ESI) 642 (M + H) ⁺ , starting from intermediates 7 and 2.2.

To a solution of a compound of formula 8.1 (4.45 g, 6.9 mmol) in 60 ml of dichloromethane at 0 ° C, 60 ml of trifluoroacetic acid are added, the mixture is stirred for 15 hours at room temperature, evaporated in vacuo, dissolved in 250 mL of DCM washed with 300 ml of 5% NaHC0 ₃ , dried over Na ₂ S0 ₄ and evaporated in vacuo. Recrystallized from a mixture of ethyl acetate with methyl? Re / i-butyl ether (1: 1). Obtain 2.7 g (71%) of cyclobutyl (5 -2 - ((5) - (((2?, ZL, 4L, 5 /?) - 5- (2,4-dioxo-3,4-dihydropyrimidin-1 ( 2 /) -yl) -3-hydroxy-4-methyl-4-fluoro-tetrahydrofuran-2-yl) methoxy) - (phenoxy) -phosphorylamino) -propanoate of the formula (1.1) as a white crystalline substance. LC-MS ( ESI) 542 (M + H) ⁺ . Ή NMR (400 MHz, DMSO-s / ₆ ) δ 1 1.51 (brs, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.38 (m, 2H), 7.23 (m, 2H), 7.19 (m, 1 H), 6.03 (m, 2H), 5.84 (d, J = 6.8 Hz, 1H), 5.55 (dd, J, = 8.0 Hz, J ₂ = 1.2 Hz, 1H), 4.85 (p, J = 7.2 Hz, 1H), 4.37 (m, IH), 4.27 (m, 1H), 4.01 (m, 1H), 3.83 (m, 2H), 2.23 (m, 2H), 1.95 (m, 2H), 1.71 (m, 1H), 1.56 (m, 1H), 1.25 (d, J = 22.8 Hz, 3H), 1.23 (d, J = 6.8 Hz, 3H). Recrystallization of a prodrug of Formula 1.1 from various solvents results in polycrystalline or crystalline forms. Recrystallization from a mixture of ethyl acetate with methyl / yaret-butyl ether (1: 1), ethanol, ethyl acetate and a mixture of acetic acid with water gives a prodrug of formula 1.1 in polycrystalline forms, which mainly include a rhombic phase with unit cell parameters a = 28.1056 (8 ) A, b = 16.8998 (4) A, c = 5.25380 (12) A and the monoclinic phase with unit cell parameters a = 16.2770 (6) A, b = 16.91 17 (8) A, c = 5.20429 (15) A, c = 1 17.822 (2) °.

 Upon recrystallization of a prodrug of formula 1.1 from a mixture of dimethyl sulfoxide with water, a substance is obtained in a white crystalline form consisting of a rhombic phase with unit cell parameters a = 28.1056 (8) A, b = 16.8998 (4) A, c = 5.25380 (12) A.

 The solubility of crystalline and polycrystalline forms after recrystallization from various solvents at pH 2 and pH 7 is close and is 0.18 - 0.25 mg / ml. The exception is a polycrystalline sample obtained by recrystallization from dimethyl sulfoxide, the solubility of which is slightly higher and has a value of 0.63 - 0.67 mg / ml (Table 1).

 Table 1. Kinetic solubility of polycrystalline and crystalline forms of prodrugs of formula 1.1 at a wavelength of 260 nm.

Prodrug of Formula Form Solubility, mg / ml

 1.1 recrystallized prodrugs at pH 2 at pH 7

 from formula 1.1 SD value SD value

Mixtures of ethyl acetate with methyl polycrystal.

 0.25 0.00 0.24 0.001 wpe / i-butyl ether

 Ethanol Polycrystal. 0.20 0.00 0.20 0.003

Ethyl Acetate Polycrystal. 0.22 0.00 0.22 0.002

Acetic Acid Polycrystal. 0.63 0.009 0.67 0.041

Dimethyl Sulfoxide Crystal. 0.18 0.00 0.18 0.003 Cyclobutyl (5) -2 - ((i?) - (((2i?, 3i?, 4i?, 5i?) - 5- (2,4-AHOKCo-3,4-dihydropyrimidin-1 (2 ^ -yl) -3-hydroxy-4-methyl-4-fluoro-tetrahydrofuran-2-yl) methoxy) - (phenoxy) -phosphorylamino) -propanoate (1) in 58% yield. LC-MS (ESI) 542 and cyclobutyl (5) -2 - ((L) - (((2L, 3L, 4β, 5 / g) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2) -yl) -3 -hydroxy-4-methyl-4-fluoro-tetrahydrofuran-2-yl) methoxy) - (phenoxy) -phosphorylamino) -propanoate (1.2) in 64% yield. LC-MS (ESI) 542 (M + H) ⁺ . Ή NM? (300 MHz, DMSO - </ ₆ ) δ 1 1.53 (brs, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.38 (m, 2H), 7.19 (m, 3H), 6.08 ( m, 2H), 5.91 (d, J = 6.3 Hz, 1H), 5.57 (d, J = 8.1 Hz, 1H), 4.85 (p, J = 7.5 Hz, 1H), 4.41 (m, 1H), 4.27 ( m, 1H), 4.05 (m, 1H), 3.79 (m, 2H), 2.23 (m, 2H), 1.94 (m, 2H), 1.7 0 (m, 1H), 1.56 (m, 1H), 1.24 (d, J = 23.4 Hz, 3H), 1.21 (d, J = 6.0 Hz, 3H).

 Example 2. Obtaining an antiviral composition in the form of tablets. Starch (500 mg), ground lactose (800 mg), talc (200 mg), and 1500 mg of a prodrug of formula 1.1 were mixed together and pressed into a bar. The resulting bar was crushed into granules and sieved through a sieve to collect 14-16 mesh granules. The granules thus obtained were formed into tablets of a suitable form weighing 400 or 800 mg each.

 Example 3. Obtaining an antiviral composition in the form of capsules. A prodrug of formula 1.1 was thoroughly mixed with lactose powder in a ratio of 1: 1. The resulting powder mixture was packaged in suitable gelatin capsules of 200 mg and 400 mg in each capsule.

 Example 4. Obtaining an antiviral composition in the form of compositions for intramuscular, intraperitoneal or subcutaneous injection. 500 mg of a prodrug of formula 1.1, 300 ml of chlorobutanol, 2 ml of propylene glycol and 100 ml of water for injection were mixed. The resulting solution was filtered, placed in ampoules of 5 ml and sealed.

Example 5. Determination of anti-HCV activity and cytotoxicity of compositions comprising prodrugs of the formula 1.1 and Sovaldi ^® . To determine the antiviral activity of the tested compositions, including prodrugs, a Huh7 human hepatocellular carcinoma cell line stably transfected with hepatitis C virus replicon (HCV) was used. 50 μl of cell suspension in complete culture medium (DMEM IX, Cellgro; cat. # 10-013-CV) was transferred to 96- well plates with a total density of 7500 cells per well. Serial dilution of the tested prodrugs was prepared from a freshly prepared 200-fold stock solution in DMSO with AND concentration points with a dilution step of 3 from 20 nM in complete medium and used as 2-fold solutions. Not less than 4 hours after planting, 50 μl of serial dilutions of prodrugs were added. The final concentration of prodrugs ranged from 10 nM to 0.1 pM, and DMSO - 0.5%. The plate with cells was incubated for 3 days at 37 ° C in a humidified atmosphere of 5% C0 ₂ . After incubation, the medium was removed by inverting the plate with gentle shaking. Cells were fixed with 100 μl of a 1: 1 solution of acetone: methanol for 1 minute, washed 3 times with phosphate buffer (PBS) and then blocked with 150 μl / well of 10% bovine fetal serum (FBS) in PBS or for one hour at room temperature. Then the cells were washed 3 times with PBS and incubated for 2 hours at 37 ° C with 100 μl / well of antibodies to non-structural protein 5B (NS5B) HCV (Affinity BioReagents; cat. # MA1-080, stock solution (1 mg / ml) was diluted 1: 4000 in 10% FBS-PBS). Cells were washed 3 times with PBS, 100 μl / well of OPD solution appeared (per 1 plate: 1 tablet of OPD dissolved in 12 ml of citrate / phosphate buffer with 5 μl of 30% H ₂ 0 ₂ ) for 30 minutes in the dark at room temperature. The reaction was stopped with 100 μl / well of 2N H ₂ S0 ₄ , after which OD490 was measured using a multifunctional reader Victor V 1420 (Perkin Elmer). EC ₀ values of the tested prodrugs were determined by plotting the activity curve in the GraphPad Prizm program. In particular, in relation to the lb (gTlb) HCV genotype in 10% bovine fetal serum (10% FBS) medium under comparable conditions, Sovaldi ^® has an EC of ₅₀ 45.0-170.0 pM and an EC90 of 520.0.0 pM, and the new prodrug of formula 1.1 has an EC ₅₀ 15.0-27.0 pM and EC 0 128.0 pM (Table 2). Thus, the new prodrug of formula 1.1 is more than three times more active than Sovaldi ^® .

Table 2. Inhibitory activity of prodrugs 1.1 and Sovaldi ^® in relation to gTlb NS5B HCV.

 10% FBS

 Prodrug

EC ₅₀ , PM EC90, PM CC90, μΜ

Formulas 1.1 15.0-27.0 128.0> 100 ^► Sovaldi '45.0-170.0 * 520.0 **> 100 **

 according to * http // www.hcvdruginfo.ca / HCV_Sofosbuvir.pdf;

 ** M. J. Sofia et al. J. Med. Chem. 2010, 53, 7202-7218.

The cytotoxicity of the tested compositions, including prodrugs, was determined in parallel on the same Huh7 cell line using the ATPLite kit (Perkin-Elmer, Boston, USA) in accordance with the manufacturer's instructions. 50 μl of cell suspension in complete culture medium (DMEM IX, Cellgro; cat. # 10-013-CV) was transferred into 96-well plates with black walls and a transparent bottom with a total density of 7500 cells per well. After 18 hours after cell planting, 50 μl of serial dilutions of the compounds were added. The plate with cells was incubated for 4 days at 37 ° C in a humidified atmosphere of 5% C0 ₂ . The cells were then washed 2 times with 200 μl / well of PBS and lysed by adding 50 μl / well of lysis buffer (all reagents from the ATPLite kit). After stirring for 5 minutes on a shaker, 50 μl / well of substrate was added. After an additional 5-minute incubation, the plate was stored for 10 minutes in the dark and the luminescence in the wells was read using a multifunctional reader Victor ³ V 1420 (Perkin Elmer). The SS ₅₀ values of the tested compounds were determined by constructing a cytotoxicity curve in the GraphPad Prizm program. In particular, for a composition comprising a prodrug of formula 1.1 and Sovaldi ^® , the values of CC ₅₀ > 100 μM (Table 2) and therapeutic window (therapeutic index TI = EC ₅ о / CC ₅ о) TI> 6,000.0

 Example 6. Determination of the kinetic solubility of substances.

The principle of the method. The studied substance is dissolved in DMSO to a concentration of 10 mM, then introduced into an aqueous solvent (phosphate buffer, water, universal buffers with different pH) to a concentration of 200 μM. The resulting solution was incubated for one hour at room temperature on a shaker in a 96-well filter plate (Millipore's MultiScreen Solubility Filter Plate), after which the precipitate was filtered off under vacuum. The absorption spectrum of the substance is recorded on a spectrophotometer in the range of 240-400 nm in increments of 10 nm. For quantitative solubility calculations, use a calibration curve of standard solutions (0 - 200 μM) with a content of 40% acetonitrile. The range of detectable concentrations is 3-200 μM. Testing is carried out in duplicates.

 Preparation of calibration standards. Calibration standards were prepared from 50-fold stock solutions in DMSO, followed by dilution in a buffer with 40% acetonitrile, which was added to ensure complete solubility of the test compound in the calibration sample. 6 standard samples with concentrations of 0.13.125, 12.5, 50, 100 and 200 μM were prepared in the wells of a 96-well UV plate by adding 4 μl of the corresponding 50x stock solutions in DMSO to 196 μl of buffer with 40% acetonitrile content. In this case, the concentration of DMSO at all points remained constant and was equal to 2% (v / v).

 To construct the calibration curves, we took the optical spectrum of a UV tablet in the wavelength range from 250 nm to 400 nm with a step of 10 nm. From the obtained spectra, for each compound, wavelengths were selected that met the following criteria:

 - At minimum substance concentration OD> 0.1 (AU)

 - At maximum substance concentration OD <2.0

 A calibration curve was constructed for each compound according to the OD dependence at the selected wavelength versus concentration.

 Determination of the kinetic solubility of compounds. Solubility was determined in a MultiScreen Solubility filter plate (Millipore Corp.) as follows: 196 μl buffer (without acetonitrile) and 4 μl JMM substance in DMSO or 4 μl DMSO (for a blank sample) were added to the well of a MultiScreen Solubility filter plate. The plate was incubated for one hour on a shaker (400 rpm) at room temperature. After incubation, the solutions were filtered through a filter plate using vacuum (10 "Hg) in a polypropylene plate with a U-shaped bottom.

 From a U-shaped bottom plate, 120 μl / well of filtrate was transferred to a new UV plate, after which 80 μl / well of acetonitrile was added.

 The optical density of the obtained solutions was measured at the previously selected wavelength for each compound.

The calculations. The final concentration of the substance in the filtrate was calculated by the formula: Cf _NLTt = (OD ^ Filtrate-AxBlankySlope х 1.67, Where:

 - ODxFiltrate - the optical density of the filtrate at a selected wavelength,

 - ODxBlank - OD idle sample,

 - Slope - slope of the calibration line,

 - 1.67 - dilution ratio of the filtrate with acetonitrile.

 The results obtained are presented in Table 1.

 Example 7. X-ray powder studies of samples of prodrugs. All diffraction patterns were recorded on a Bruker D8 Advance Vario diffractometer,

equipped with an x-ray tube with a copper anode and a Ge (l 1 1) -monochromator (CuKo ^A ) and a position-sensitive detector LynxEye, in installations in the light. The shooting interval was 3-90 ° 26 for sample s5 and 5.7-90 ° 26 for the rest of the samples, step 0.01 ° 26. The analysis was performed using the Bruker Topas5 program ['Bruker TOPAS 5 User Manual. — Karlsruhe, Germany: Bruker AXS GmbH, 2015 .].

Samples obtained by recrystallization of 1B.7 from a mixture of ethyl acetate with methyl / and et-butyl ether (1: 1), ethanol, ethyl acetate and a mixture of acetic acid with water have a polycrystalline form. Powder X-ray phase studies of these samples show that the samples have the same qualitative phase composition and slightly differ in phase ratio. The samples contain a rhombic phase with unit cell parameters a = 28.1056 (8) A, b = 16.8998 (4) A, c = 5.25380 (12) A. An analysis of systematic extinctions suggests the spatial group Ρ2ι2] 2ι. The unit cell volume 2495.45 (1 1) A ³ corresponds to the declared composition and Z '= 1. The samples also contain a monoclinic phase with unit cell parameters a = 16.2770 (6) A, b = 16.91 17 (8) A, c = 5.20429 (15 ) A, b = 1 17.822 (2) °. Systematic extinctions suggest spatial group P2). The unit cell volume of 1266.98 (9) A ³ corresponds to the declared composition and Z '= 1. An estimate of the phase ratio, based on a comparison of the integral intensities of the peaks, predicts the content of the monoclinic phase from 30 to 50%.

The sample obtained by recrystallization of 1B.7 from a mixture of dimethyl sulfoxide with water is a white crystalline solid. A sample of this form, according to powder x-ray studies, is single-phase and consists of a rhombic phases with unit cell parameters а = 28.1056 (8) А, b = 16.8998 (4) А, с = 5.25380 (12) А. An analysis of systematic extinctions suggests the space group Ρ2ι2ι2ι. The unit cell volume 2495.45 (11) A ³ corresponds to the claimed composition and Z '= 1.

 Example 8. Determination of stability in biological environments of compositions comprising prodrugs of the formula 1.1. The starting compositions comprising prodrugs of formula 1.1 (test compound) were prepared at a concentration of 10 mM in DMSO, from which 100-fold working solutions were then prepared at a concentration of 100 μM in a mixture of acetonitrile: water with a volume ratio of 1: 1.

a) Stability in the S9 fraction. The reaction mixture was prepared in 0.1 M potassium phosphate buffer (pH 7.4 BD Gentest) in a total final volume of 250 μl and contained 1 mM NADPH tetrasodium salt (AppliChem), 7 mM glucose-6-phosphate sodium salt (Sigma) , 1.5 U / ml glucose-6-phosphate dehydrogenase (Sigma), 3.3 mM MgCl ₂ (Sigma), 5 mM uridine-5-diphosphate-glucuronic acid trisodium salt (UDFGK, Sigma) and 1 μM test compound (final concentrations indicated) . The metabolic reaction was initiated by adding a suspension of S9 human liver fractions (BD Gentest), the final protein concentration was 1 mg / ml. The reaction mixture was incubated at 37 ° C on a shaker (Vortemp56) with stirring at 400 rpm. At certain intervals (0, 0.25, 0.5, 1, 2, 4, 6, 8, 24 h), 30 μl of sampling was carried out, the reaction was stopped by adding 180 μl of cold acetonitrile containing an internal standard to the selected sample. Protein precipitation was carried out on ice for 15 minutes. After that, the samples were centrifuged for 10 min at 3000 rpm. 150 μl of the supernatant was selected for analysis. Incubation was carried out in two repetitions, each sample was measured twice.

b) Stability in artificial gastric and intestinal juices. A test composition comprising a prodrug of formula 1.1 at a final concentration of 1 μM was incubated in artificial gastric juice (0.2% NaCl in 0.7% v / v HC1) and artificial intestinal juice (0.05 M KH ₂ P0 ₄ , pH b) 6 , 75). Incubation was carried out in a shaker incubator (Vortemp56) at 37 ° C and stirring at 300 rpm. At certain intervals (0, 0.25, 0.5, 1, 2, 4, 6, 8, 24 h), sampling was carried out in 30 μl, the reaction was stopped by adding 180 μl of cold acetonitrile containing the internal standard to the sample. After that, the samples were centrifuged for 10 min at 3000 rpm. 150 μl of the supernatant was selected for analysis. Incubation was carried out in two repetitions, each sample was measured twice.

 c) Stability in blood plasma. A test compound at a final concentration of 1 μM was incubated in pooled human blood plasma (Innovative Research). Incubation was carried out in a shaker incubator (Vortemp56) at 37 ° C and stirring at 300 rpm. At certain intervals (0, 0.25, 0.5, 1, 2, 4, 6, 8, 24 h), 30 μl of sampling was carried out, the reaction was stopped by adding 180 μl of cold acetonitrile containing an internal standard to the selected sample. After that, the samples were centrifuged for 10 min at 3000 rpm. 150 μl of the supernatant was selected for analysis. Incubation was carried out in two repetitions, each sample was measured twice.

Sample Analysis. Samples were analyzed by HPLC-MS / MS, developed for each prodrug tested, using a 1290 Infinity II chromatographic system (Agilent Technologies) coupled to a QTRAP5500 tandem mass spectrometer (AB Sciex). When developing conditions for mass spectrometric detection, solutions of the tested compounds in a 1: 1 acetonitrile-water mixture with a concentration of 100 ng / ml were analyzed by direct injection into the mass spectrometer using a syringe pump during electrospray ionization in the mode of registration of positive ions. When scanning in the full ion current mode (MS1), the molecular ion for each compound was determined, the main product ions were recorded in the MS2 mode. Then, the MS / MS method was optimized in the MRM mode to achieve maximum sensitivity. In the quantitative processing of chromatograms, the most intense MRM transition was used for the analyte and the internal standard. Chromatographic separation was performed on a YMC Triart C 18, 50x2 mm, 1.9 μm column in a gradient elution mode in the mobile phase with a composition of 0.1% formic acid in water - 0.1% formic acid in acetonitrile. Tolbutamide (Fluka) was used as an internal standard. W 201

33

The calculations. The kinetics of the decrease of the tested prodrug in the antiviral composition during incubation in the biological medium was calculated half-life (T1 2). For the calculations, the values of the areas of chromatographic peaks of substances normalized to the signal of the internal standard were used in the experimental samples. Using the linear dependence of log-normalized areas of chromatographic peaks on time, we calculated the elimination rate constant k, the slope of the linear section). Next, the half-life was calculated: Tm = 0.693 / k. In particular, it was found (Table 3) that the prodrug of formulas 1.1, 1.2 and Sovaldi® in the antiviral composition have comparable stability in human gastric juice (Ti / ₂ ^SGF = 12.7 h - 15 h), in human intestinal juice (Ti ₂ ^SIF > 24 h) and in human plasma (Τι _/ ^ ¹ > 24 h). At the same time, a prodrug of formula 1.1 metabolizes more quickly in the S9 fraction of human liver microsomes and has a half-life of T = 0.05 h, while its prototype Sovaldi®HMeeT Ti _{/ 2} ^HS9 = 0.54 h (Table 3), i.e. . a prodrug of formula 1.1 is 1 to 1 times faster metabolized in the S9 fraction of human liver microsomes than Sovaldi®.

Table 3. The stability and activity of antiviral compositions, including a new prodrug of the formula 1.1 and Sovaldi ^®

Example 9. The study of the pharmacokinetics (PK) of compositions comprising a prodrug of formula 1.1 and Sovaldi® in rat liver.

Preparation of compositions comprising a prodrug of formula 1.1 and Sovaldi® for administration to rats. The test composition was administered at a dose of 50 mg / kg. For this purpose, compositions were prepared with a prodrug concentration of the formula 1.1 or Sovaldi ^® _5.0 mg / ml in 0.5% hydroxypropyl methylcellulose (HPMC) with the addition of 5% ethanol as follows: The required amount of HPMC was added to a sample of a prodrug of Formula 1.1 or Sovaldi and ground in a mortar, then the required amount of 5% ethanol in distilled water was gradually added portionwise, mixed thoroughly until a suspension suitable for intragastric administration was obtained.

Administration of compositions comprising a prodrug of Formula 1.1 and Sovaldi ^® to animals. Obtaining samples of blood plasma and liver. The study was conducted on laboratory rats of the Sprague Dawley line. Rats were divided into groups, according to the selected time points (1, 2, 4, 6, 8, 10, 12, 16 and 24 hours), 6 animals in the group. The animals were weighed, the volume of the administered composition, including the prodrug of formula 1.1 or Sovaldi ^® , was calculated for each animal at a rate of 10 ml / kg. The introduction of compositions comprising a prodrug of formula 1.1 or Sovaldi ^® , was carried out intragastrically through a tube. Between injections, animals at one time point were spaced sufficiently to take a liver and blood sample. After the desired period of time after administration of rat euthanized by inhalation of C0 _2. Immediately after euthanasia, the animal was opened as quickly as possible, the upper lobe of the liver was cut off and immediately thrown into liquid nitrogen. A frozen liver fragment was placed in a labeled tube cooled in liquid nitrogen. Until the end of the experiment, the samples were stored in liquid nitrogen, then transferred to a -80 ° C low-temperature freezer.

Sample preparation. A sample of a liver weighing about 1 g was ground in a mortar while cooling with liquid nitrogen, the resulting powder was poured into a three-fold volume of methanol with 70% methanol with EDTA and homogenized using an Omni Bead Ruptor 24 homogenizer at a speed of 6.3 m / s for 45 sec 2 times with an interval of 10 sec . 40 μl of a ten-fold standard solution containing PSI-7409 and Н027-4261 (or methanol in the case of experimental samples) and 100 μl of an internal standard solution (5-bromuridine triphosphate) with a concentration of 25 t ng / ml were added to 360 μl of the obtained homogenate. After stirring and centrifugation, 400 μl of the supernatant was diluted with 400 μl of a solution of 1% formic acid in a methanol-water mixture (1: 1). Solid phase extraction was performed on Waters Oasis WAX cartridges, 800 μl of a 5% solution of ammonia in methanol was eluted, the eluate was evaporated and re-dissolved in 200 μl of methanol. Conditions for HPLC-MS / MS analysis. The analysis was performed by HPLC-MS / MS using an Agilent 1290 Infinity II HPLC system coupled to an AB Sciex QTrap 5500 mass spectrometer. Separation was performed on a Thermo Hypercarb column (50x3mm, 5 μm), solution 25 was used as the mobile phase A (PFA) 25 mM ammonium acetate with the addition of 0.5% ammonia; as mobile phase B (PFB), a solution of 25 mM ammonium acetate in a mixture of water-isopropanol-acetonitrile (1: 1: 3) with the addition of 0.5% ammonia. The separation was carried out in a gradient mode: 0-0.3 min - 5% PFB; 3-3.4 min - 50% PFB; 3.6-4.5 min - 5% PFB, PSI-7409 and Н027-4261 were recorded in the MRM mode using ion transitions 499/159 and 410/150, respectively.

Pharmacokinetic analysis. Pharmacokinetic analysis of liver-time concentration data was performed using a model-independent method using the Phoenix ™ WinNonlin® 6.3 software (Pharsight Corp.) and the GraphPad Prizm software. The following pharmacokinetic parameters were calculated: maximum concentration in the liver (C _max ) and time to reach it (T _max ), half-life ( _\ n), area under the FC curve (AUCo-t, AUCo-inf). The data obtained are presented in Table 4. As can be seen from Table 4, the concentration and AUC of _{24 h of} PSI-7409 triphosphate formed during the metabolism of a prodrug of formula 1.1 in rat liver is Stax = 3.224.0 ng / g and AUC _{24 h} = 30.487.0 ng h / g, while with a similar metabolism, Sovaldi ^® has Stax = 1, 934.0 ng / g and AUC _{24 h} = 16,796.0 ng h / g {Table 4). This suggests that the new prodrug is almost 2 times more efficiently metabolized in the liver to the desired triphosphate PSI-7409 (medicine).

Table 4. RK parameters triphosphate PSI-7409 in rat liver after oral administration of the prodrug of formula 1.1 and Sovaldi ^® at 50 mg / kg.

 RK parameters Processing RK results using according to M. J.

 Sofia et al. J. Med.

 Chem. 2010, 53,

Phoenix ™ WinNonlin® 6.3 GraphPad Prizm

 7202-7218.

Prodrug of Formula 1.1 Sovaldi ^®

7.2 5.5

 AUCo-inf, ng.h / g 33.823.0 18.080.0

 Industrial applicability

 The invention can be used in medicine and veterinary medicine.

Claims

Claim

1. An antiviral pharmaceutical composition for the combined treatment of hepatitis C virus in a subject in need thereof, which contains an effective amount of a prodrug selected from cyclobutyl (5) -2 - {[(2L, ZL, 4 /?, 5L) -5- ( 2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl) -4-fluoro-3-hydroxy-4-methyl-tetrahydrofuran-2-ylmethoxy] -phenoxy-phosphorylamino} -propanoate of the general formula 1, and / or cyclobutyl (5) -

2- {(5) - [(2L, 3L, 4 / ?, 5 / g) -5- (3,4-dihydro-2,4-dioxo-2H-pyrimidin-1-yl) -3-hydroxy- 4-methyl-4-fluoro-tetrahydro-furan-2-ylmethoxy] -phenoxy-phosphorylamino} -propanoate of the formula 1.1 and / or cyclobutyl (5) -2- {(R) - [(2R, 3R, 4R, 5R) -5- (3,4-dihydro-2,4-dioxo-2 ^ pyrimidin-1-yl) -

3-hydroxy-4-methyl-4-fluoro-tetrahydro-furan-2-ylmethoxy] -phenoxy-phosphorylamino} propanoate of formula 1.2, their isotopically enriched analogue, crystalline or polycrystalline form, and a pharmaceutically acceptable carrier

1.2

2. The antiviral pharmaceutical composition according to claim 1, which further comprises a therapeutically effective amount of an NS5A HCV inhibitor.

 3. The antiviral pharmaceutical composition according to claim 1, which further comprises a therapeutically effective amount of an NS3 HCV inhibitor.

 4. The antiviral pharmaceutical composition according to claim 1, which further comprises a therapeutically effective amount of an NS3 / 4A HCV inhibitor.

 5. The antiviral pharmaceutical composition according to claim 1, which further comprises a therapeutically effective amount of an HBV DNA polymerase inhibitor.

 6. The antiviral pharmaceutical composition according to claim 1, which further comprises a therapeutically effective amount of an HIV-1 reverse transcriptase inhibitor.

 7. The antiviral pharmaceutical composition according to claim 1, which further comprises a therapeutically effective amount of an HBV DNA polymerase inhibitor and an HIV-1 reverse transcriptase inhibitor.

 8. Antiviral pharmaceutical composition according to claims 1 or 2, including as an NS5A inhibitor, Daclatasvir (Daclatasvir, BMS-790052) or Hepavivir (Hepavivir, AV-4025), or AV-4067, or AV-4084, or AV-4056, or AV-4058, or Ombitasvir ( Ombitasvir, АВТ-267), or Elbasvir (Elbasvir, MK-8742) or Velapatasvir (Velpatasvir, VEL, GS-5816).

 9. Antiviral pharmaceutical composition according to claims 1 or 2, including Narlaprevir (Narlaprevir, SCH 900518) as an NS3 inhibitor.

 10. Antiviral pharmaceutical composition according to claims 1 or 2, including Simeprevir (Simeprevir, Olysio) as an NS3 / 4A inhibitor.

 1 1. A method for the combined treatment of hepatitis C in a subject in need thereof, comprising sequentially or simultaneously administering a therapeutically effective amount of an antiviral pharmaceutical composition according to claim 1.

12. A method for the combined treatment of hepatitis C in a subject in need thereof, comprising sequential or simultaneous administration of a therapeutically an effective amount of an antiviral pharmaceutical composition according to claim 1 and one or more anticancer drugs and / or another antiviral agent.

 13. The combination treatment method according to claim 1, comprising, as another antiviral agent, Daclatasvir (Daclatasvir, BMS-790052), Hepavivir (Hepavivir, AV-4025), AV-4067, AV-4084, AV-4056, AV-4058 , Ombitasvir (Ombitasvir, АВТ-267), Elbasvir (Elbasvir, MK-8742), Velapatasvir (Velpatasvir, VEL, GS-5816).

 14. The combination treatment method of Claim 1 1, comprising Narlaprevir NS3 inhibitor (Narlaprevir, SCH 900518) as another antiviral agent.

 15. The combination treatment method according to claim 1, comprising Simeprevir (Simeprevir, Olysio), an NS3 / 4A inhibitor, as another antiviral agent.

 16. The combination treatment method of claim 1, comprising an HBV DNA polymerase inhibitor as another antiviral agent.

 17. The combination treatment method according to claim 1, comprising an HIV-1 reverse transcriptase inhibitor as another antiviral agent.

 18. The combination treatment method according to claim 1, comprising an HBV DNA polymerase inhibitor and an HIV-1 reverse transcriptase inhibitor as another antiviral agent.

 19. The method of combination treatment according to p. 1 1, comprising as another agent one or more anti-cancer drugs.